Laser-scanning microscopy, confocal or multiphoton based microscopy, provides for performing optical sectioning.
The confocal microscope, disclosed in U.S. Pat. No. 3,013,467, utilizes optical sectioning of microscopic samples, i.e. the rejection of out-of-focus scattering, by using a confocal pinhole in front of the detection system.
The field of confocal microscopy relies on the idea of point-by-point illumination of a sample and using mechanical scanning (i.e. displacing the light beam and/or the sample) in order to collect an image. The imaging is thus a time consuming process, which is typically limited to a few tens of milliseconds per section in current commercial systems, a limit set by the speed of mechanical scanning devices. This is an inherent limitation of the confocal method, which significantly limits its utility for fast time-resolved imaging.
Multiphoton microscopes offer a different mechanism for optical sectioning. Here, a need for rejecting out-of-focus scattering is practically eliminated due to the nonlinear dependence of the measured signal on the illumination intensity. A multiphoton process, most commonly two-photon excitation fluorescence (TPEF), is efficient only when the peak intensity of the illuminating light is high, i.e. at the focal spot [Denk, W., Strickler, J. H., Webb, W. W., Two-photon laser scanning fluorescence microscopy, Science 248, 73-76 (1990)]. Due to the high energy densities required for inducing nonlinear processes, multiphoton microscopes require the use of a short pulsed laser. However, acquisition of an image still requires scanning of either the sample or the laser beam, resulting in a similar restriction of the image frame rate.
Numerous methods have been developed to increase the image acquisition rate in both confocal and multiphoton microscopes. Most of these methods involve multi-point illumination and scanning in a single spatial axis. Common examples are single-axis scanning and the use of line illumination [Sheppard, C. J. R., Mao, X. Q., Confocal microscopes with slit apertures, J. Mod. Optics 35, 1169-1185 (1988); Brakenhoff, G. J., Squier, J., Norris, T., Bliton, A. C., Wade, M. H., Athey, B., Real-time two-photon confocal microscopy using a femtosecond, amplified, Ti:Sapphire system, J. Microscopy 181, 253-259 (1995)]; and rotation of a patterned disk (in confocal microscopy) [Egger, M. D., Petran, New reflected-light microscope for viewing unstained brain and ganglion cells, Science 157, 305-307 (1967)] or a lenslet array (in multiphoton microscopy) [Buist, A. H., Muller, M., Squier, J., Brakenhoff, G. J., Real-time two-photon absorption microscopy using multipoint excitation, J. Microscopy 192, 217-226 (1998); Bewersdorf, J., Pick, R., Hell, S. W., Multifocal multiphoton microscopy, Opt. Lett. 23, 655-657 (1998)]. Others have used chromatic multiplexing [Tearney, G. J., Webb, R. H., Bouma, B. E., Spectrally encoded confocal microscopy, Opt. Lett. 23, 1152-1154 (1998)]. Overall, however, frame rates have not been reduced significantly beyond video-rate imaging.
The use of a rotatable microlens array in a microscope is also disclosed in Minoru Kobayasky et al. “Second-harmonic-generation microscope with a microlens array scanner”, Optics letters, Vol. 27, No. 15, Aug. 1, 2002. According to this technique, the microlens array is located in the optical path of a collimated input laser beam, and splits the laser beam into a plurality of beamlets, which are collimated again and are incident upon a water-immersion objective lens. This way, a multiple number of foci illuminating a specimen are used. Each focus spot is timely separated from adjacent spots, preventing interference between neighboring focal fields from causing degradation in 3D resolution. By rotation of the microlens array disk, each focal spot on the specimen is scanned simultaneously.
Yet another technique, disclosed in L. Saconi et al., “Multiphoton multifocal microscopy exploiting a diffractive optical element”, Optics letters, Vol. 28, No. 20, Oct. 15, 2003, utilizes a miniature diffractive optical element in tandem with galvo scanners to produce neat multispot grids with high diffraction efficiency and provides a high degree of uniformity in focal intensity. Here, several telescopic lens pairs are used for pivoting the grid on galvo scanners.
U.S. Pat. No. 6,020,591 discloses a two-photon fluorescence microscope with plane wave illumination. This technique is aimed at eliminating the need for moving parts to effect lateral scanning of the laser beam as well as creation of a three-dimensional image, in a two-photon fluorescence microscope. This microscope employs two laser beams having pulses of respective wavelengths λ2 and λ3, which cause two-photon emission of a fluorophore when the pulses are spatially and temporally overlapping. The pulses of the two beams of wavelengths λ2 and λ3 are combined at some crossing angle θ within the specimen, causing two-photon absorption within a line-shaped region during each instant of overlap. As the pulses pass through each other, the overlapping line-shaped region moves such that a slice of the fluorophore-containing specimen is excited by two-photon absorption during the overlap period. Lateral scanning is effected without moving parts by adjusting the relative delay of the pulses in the two beams. When the crossing angle θ is set to 0, i.e. when the two beams are directed along the same axis, the pulses of the two beams form a pancake-shaped volume in which two-photon excitation occurs as the pulses spatially and temporally overlap while traveling in opposite directions. A two-dimensional detector, such as a two-dimensional charge coupled device (CCD) array, can be used to detect a two-dimensional portion of the specimen at one time without lateral scanning. A three-dimensional image can be produced by adjusting the time delay between the two pulses, thereby changing the location of the “pancake” volume created by the intersection of the two pulses.
The conventionally used laser-scanning microscopy, be it confocal or multiphoton microscopy, although being capable of performing optical sectioning, requires a long image acquisition time, of an order of tens of milliseconds, due to the scanning process [Wilson, T., Confocal Microscopy, Academic press, London (1990)].